CN112725379A - Construction method and application of humanized CD40 gene modified animal model - Google Patents

Abstract

The invention provides a construction method and application of a humanized CD40 gene modified animal model, and relates to the field of genetic engineering. The invention successfully prepares a humanized model animal of the CD40 gene, the humanized CD40 protein can be normally expressed in the model body, and the humanized CD40 protein can be used for the function research of the CD40 gene and the screening and evaluation of the humanized CD40 antibody. The animal model prepared by the invention can be used for drug screening, drug effect research, immune-related diseases, tumor treatment and other applications aiming at the target site of human CD40, quickens the research and development process of new drugs, saves time and cost, and reduces the drug development risk. Provides a powerful tool for researching the function of the CD40 protein and screening tumor drugs.

Description

Construction method and application of humanized CD40 gene modified animal model

Technical Field

The invention relates to the field of genetic engineering, in particular to a construction method and application of a humanized CD40 gene modified animal model.

Background

In recent years, with the marketing of drugs aiming at immune checkpoint targets such as PD-1, CTLA4 and the like, tumor treatment is at the wind gap wave tip of the immune revolution, and the tumor immunotherapy becomes a new treatment mode after radiotherapy, chemotherapy and targeted therapy. The most successful of current tumor immunotherapy are drugs developed against immune checkpoints, which can be summarized in two main types: one is to use the targeting molecule to relieve the inhibition of the immunosuppressive check point molecule to the immune system to kill tumor cells, thereby killing the tumor; CTLA-4 antibody, PD-1 antibody, PD-L1 antibody and other drugs belong to this class. The other is to use the targeting molecule to activate the immune checkpoint activating molecule which is helpful for the immune system to kill the tumor, so that the tumor killing effect of the molecule is more effective and more durable; immune agonists belong to the class of drugs and are a new focus of tumor immunity research and development all over the world at present.

CD40 is a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily, and OX40, 4-1BB, etc., are immunostimulatory molecules and are key immune co-stimulatory receptors that are constitutively or inducibly expressed on the surface of a variety of immune and non-immune cells, including B cells, macrophages, Dendritic Cells (DCs), microglia, endothelial cells, epithelial cells, and keratinocytes. Its ligand CD40L (CD154) in activated CD4⁺T cell surface transient expression, in autoimmune diseases also in other cell types. CD40 and CD154 interact on the cell surface to promote intracellular signaling by recruiting TNFR-related factors (TRAFs) in the intracellular membrane, activating diverse signaling pathways such as the canonical and non-canonical nuclear factor kB pathways, mitogen-activated protein kinase, phosphatidylinositol-3Kinase (PI3K) and phospholipase C gamma pathway, and the like, are involved in humoral immunity and cellular immune response of the body: plays a key role in the activation, proliferation and differentiation of B cells, antibody production and Ig class switching, and also plays an important regulatory role in the activation of T cells and the secretion of effector cytokines.

The interaction between CD40 and CD154 can produce an anti-tumor immune effect by activating innate immunity and adaptive immunity: interaction between CD40 on dendritic cells and CD154 on T cells, leading to T cell activation; meanwhile, CD40 can also activate B cells and macrophages, so that neutrophils and NK cells are activated, and finally T cells are activated, so that an anti-tumor effect is generated. In addition, the interaction of CD40-CD154 is a target of other immune-related diseases besides tumor immunotherapy, and it has been reported that the interaction of CD40-CD154 is regulated by gene or antibody intervention means, and has a certain therapeutic effect on arteriosclerosis RA, Crohn's disease, encephalomyelitis (EAE), and the like in mouse models.

In view of the important role of the CD40 target in innate immunity and adaptive immunity, according to incomplete statistics, more than 25 medicaments aiming at the CD40 target have been clinically introduced, wherein 15 medicaments of Nowa, Baishigui, Erberweiqi and other medicament enterprises are already in the clinical stage II, and the related indications comprise tumors such as glioma, non-small cell lung cancer, gastric cancer, melanoma and the like, as well as autoimmune system diseases such as psoriasis, arthritis, Crohn's disease and the like. Thus, the CD40 target has become a hot target for tumor immunotherapy and the treatment of autoimmune diseases.

The homologous gene of the human CD40 gene in a mouse is Cd40, the two genes have similar gene structures, but in the aspect of amino acid consistency, the amino acid consistency of human and mouse CD40 proteins is only 61%, the amino acid sequence consistency of extracellular regions is only 59%, and the difference in the amino acid sequence consistency probably causes that a medicament aiming at the human CD40 target point cannot identify the Cd40 target point of the mouse, and the in-vivo efficacy evaluation of the medicament by using a wild mouse cannot be carried out. In addition, although immunotherapy has a certain effect on cancer treatment, if the immune response is too strong, it causes great damage to healthy tissues and causes immunotoxic side effects, and therefore, strict screening of the test drugs in terms of safety of toxicity and the like is required in addition to evaluation of drug efficacy. However, in the conventional wild-type mice, because of differences in human and mouse targets, the drug cannot identify the target in the body of the mouse, and is not suitable for toxicity and safety evaluation. In order to reduce the difference between human and mice, the wild type mouse is humanized and transformed, so that the microenvironment of a human body can be well reproduced, and the method can be applied to drug toxicity and safety evaluation while being used for drug effect evaluation. At present, humanized mouse models have been widely used in the field of disease research such as tumor and virus infection.

Disclosure of Invention

The target molecules are humanized and transformed by means of gene modification, and the problem of target difference between human and animals can be well solved by establishing a humanized animal model to research human diseases: after gene modification, the humanized model animal body can fully or partially express corresponding human protein, imitate the interaction between various molecules when human diseases occur, reappear the characteristics of the human diseases, greatly reduce the difference between the animal model and the human body before clinic, and enable the screening research of the medicine at the whole level of the animal to be possible; meanwhile, the model can evaluate the toxicity and drug metabolism of the drug on a model with a normal immune system, can better fit the clinical drug efficacy, and improves the success probability of drug screening.

The invention aims to provide a construction method of a humanized CD40 gene modified animal cell, a humanized CD40 gene modified animal cell, a construction method of a humanized CD40 gene modified animal model and application of a humanized CD40 gene modified animal model prepared by the method, a cell line or a cell culture, a tissue or an organ derived from the humanized CD40 gene modified animal model or filial cells or cell lines or cell cultures thereof, application of the cell, the cell line or the cell culture, the tissue or the organ, a chimeric CD40 protein, a chimeric CD40 gene encoding the chimeric CD40 protein, a recombinant vector containing the chimeric CD40 protein, a cell containing the recombinant vector and a tissue containing the cell, so as to accelerate research progress of fields related to the human CD40 gene or protein.

In order to achieve the above object, the present invention firstly provides a method for constructing a humanized CD40 genetically modified animal cell, comprising:

replacing the sequence of the No. 2 to No. 5 exons of the non-human CD40 gene in the non-human animal cell with the sequence of the No. 2 to No. 5 exons of the human CD40 gene to form a humanized CD40 gene, so as to obtain a humanized CD40 gene modified animal cell;

preferably, the non-human animal is a rodent; more preferably, the rodent is a mouse;

preferably, the cell is a fertilized egg.

Specifically, the sequence of exons 2 to 5 of the murine Cd40 protein is the extracellular domain.

Further preferably, the mouse is a C57BL/6 mouse.

In some embodiments, the method for constructing the humanized CD40 genetically engineered animal cell comprises:

a mixture of a human CD40 gene homologous recombination vector, sgRNA, and Cas9, the Cas9 including Cas9mRNA and/or Cas9 protein, is provided and introduced into the non-human animal cell.

Specifically, the mixture may be introduced into the non-human animal cell by microinjection.

In some embodiments, the 5 'target site of the sgRNA is located in intron 1 of the mouse Cd40 gene and the 3' target site of the sgRNA is located in intron 5 of the mouse Cd40 gene.

Preferably, the sequence of the 5' end target site targeted by the sgRNA is as shown in SEQ ID NO: 18-27, the sequence of the sgRNA-targeted 3' end target site is as shown in SEQ ID NO: 28-37;

more preferably, the sequence of the 5' target site targeted by the sgRNA is as set forth in SEQ ID NO: 22, the sequence of the sgRNA-targeted 3' end target site is shown as SEQ ID NO: shown at 36.

In some embodiments, the human CD40 gene homologous recombination vector comprises a 5 'homology arm, a human CD40 gene fragment, and a 3' homology arm arranged sequentially from 5 'end to 3' end;

preferably, the sequence of the human CD40 gene fragment is shown in SEQ ID NO: 11 is shown in the figure;

preferably, the sequence of the 5' homology arm is as shown in SEQ ID NO: 9, the sequence of the 3' homology arm is shown as SEQ ID NO: shown at 10.

In some embodiments, the humanized CD40 gene is selected from at least one of the following groups:

(a) the CDS coding sequence of the humanized CD40 gene is shown as SEQ ID NO: 6 is shown in the specification;

(b) the mRNA sequence transcribed by the humanized CD40 gene is shown as SEQ ID NO: 7 is shown in the specification;

(c) the protein sequence coded by the humanized CD40 gene is shown as SEQ ID NO: shown in fig. 8.

The invention also provides a humanized CD40 gene modified animal cell, wherein the humanized CD40 gene modified animal cell is obtained by the construction method of the humanized CD40 gene modified animal cell.

The invention also provides a construction method of the humanized CD40 gene modified animal model, which comprises the steps of transplanting the humanized CD40 gene modified animal cell or the embryo obtained by the development of the humanized CD40 gene modified animal cell into a surrogate mother body for development to obtain an F0 animal;

f0 generation animals are tested to obtain positive animals with correct genotype identification of F0 generation.

In some embodiments, the method for constructing the humanized CD40 genetically modified animal model further comprises: and (3) expanding the population quantity of the F0 generation positive animals in a hybridization and selfing mode, selfing the heterozygote animals to obtain homozygote animals, and establishing a stable humanized CD40 gene modified animal strain.

The invention also provides a cell, a cell line or a cell culture, a tissue or an organ, wherein the cell, the cell line or the cell culture, the tissue or the organ is derived from a humanized CD40 gene modified animal model or filial generation thereof, and the humanized CD40 gene modified animal model is obtained by the construction method of the humanized CD40 gene modified animal model.

In particular, the tissue or organ may be spleen, a tumor or a culture thereof.

The invention also provides a humanized CD40 gene modified animal model obtained by the construction method of the humanized CD40 gene modified animal model, and application of cells, cell lines or cell cultures, tissues and organs derived from the humanized CD40 gene modified animal model in the fields related to human CD40 genes or proteins;

optionally, the application comprises at least one of human CD40 gene function research, human CD40 antibody research, medicine preparation aiming at a human CD40 target site and drug effect research.

Preferably, the applications include the development of products requiring immune processes involving human cells, the manufacture of human antibodies, or as model systems for pharmacological, immunological, microbiological and medical studies or the production of immune processes involving human cells and the use of animal experimental disease models for the production of immune processes involving human cells, for the research of etiology and/or for the development of new diagnostic and/or therapeutic strategies or for in vivo studies, screening of human CD40 signaling pathway modulators, pharmacodynamic testing, screening libraries, efficacy assessment, screening, validation, evaluation or research of CD40 gene function studies, human CD40 antibodies, drugs directed against human CD40 target sites, pharmacodynamic studies, immune-related disease drugs and anti-tumor drugs.

The invention also provides a chimeric CD40 protein, wherein the chimeric CD40 protein is selected from one of the following groups:

a) the sequence of the chimeric CD40 protein is SEQ ID NO: 8;

b) the sequence of the chimeric CD40 protein is similar to that of SEQ ID NO: 8 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%;

c) the sequence of the chimeric CD40 protein is similar to that of SEQ ID NO: 8 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid;

d) the sequence of the chimeric CD40 protein is represented by SEQ ID NO: 8 by substitution and/or deletion and/or insertion of one or more amino acid residues and has the same function.

Specifically, substitution and/or deletion and/or insertion of one or more amino acid residues means substitution and/or deletion and/or insertion of not more than ten amino acid residues.

The invention also provides a chimeric CD40 gene for encoding the chimeric CD40 protein, wherein the chimeric CD40 gene is selected from one of the following groups:

a) the CDs sequence of the chimeric CD40 gene is SEQ ID NO: 6;

b) the mRNA sequence of the chimeric CD40 gene is SEQ ID NO: 7;

c) the CDS sequence of the chimeric CD40 gene is similar to that of SEQ ID NO: 6 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%;

d) the CDS sequence of the chimeric CD40 gene is similar to that of SEQ ID NO: 6 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide;

e) the CDs sequence of the chimeric CD40 gene has the amino acid sequence set forth in SEQ ID NO: 6 by substitution and/or deletion and/or insertion of one or more nucleotides;

f) the part of the chimeric CD40 gene derived from the human CD40 gene is SEQ ID NO: 11;

g) the part of the chimeric CD40 gene, which is derived from the human CD40 gene, is similar to the sequence shown in SEQ ID NO: 11 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%;

h) the part of the chimeric CD40 gene, which is derived from the human CD40 gene, is similar to the sequence shown in SEQ ID NO: 11 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide;

i) the part of the chimeric CD40 gene which is derived from a human CD40 gene is a nucleotide sequence which is formed by the amino acid sequence shown in SEQ ID NO: 11 by substitution and/or deletion and/or insertion of one or more nucleotides.

Specifically, substitution and/or deletion and/or insertion of one or more nucleotides means substitution and/or deletion and/or insertion of not more than ten nucleotides.

The invention also provides a recombinant vector containing the chimeric CD40 gene.

Specifically, the recombinant vector may be a cloning vector or an expression vector.

The invention also provides a cell containing the recombinant vector.

The invention also provides a tissue comprising the above cells.

The invention has the beneficial effects that:

the invention successfully prepares a humanized model animal of the CD40 gene, the humanized CD40 protein can be normally expressed in the model body, and the humanized CD40 protein can be used for the function research of the CD40 gene and the screening and evaluation of the humanized CD40 antibody.

The animal model prepared by the invention can be used for drug screening, drug effect research, immune-related diseases, tumor treatment and other applications aiming at the target site of human CD40, quickens the research and development process of new drugs, saves time and cost, and reduces the drug development risk. Provides a powerful tool for researching the function of the CD40 protein and screening tumor drugs.

Furthermore, the humanized CD40 mouse constructed by the invention replaces the extracellular region of the mouse Cd40 gene with a human sequence, and the intracellular region retains the complete mouse sequence. The humanized mouse which is successfully made owns the human extracellular region, the humanized CD40 target drugs can be screened, while the mouse intracellular region ensures that the intracellular signal transduction is not influenced, and the external stimulation is faithfully transformed into the intracellular behaviors.

Drawings

To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, and it should be understood that the following drawings only illustrate some embodiments of the present invention, and therefore should not be considered as limiting the scope of the present invention.

FIG. 1 is a schematic diagram showing the comparison between murine Cd40 and human CD40 genes.

FIG. 2 is a schematic diagram of a humanized mouse Cd40 gene after modification.

FIG. 3 is a schematic diagram of a targeting scheme for humanizing a mouse Cd40 gene.

FIG. 4 is a schematic diagram showing the cleavage result in example 4 of the present invention; 1: as a result of XbaI enzyme digestion identification, the theoretical band size should be 6.3kb, 4.2kb, 3.3kb and 2.3 kb; m: 1kb DNA ladder.

Fig. 5A shows the results of activity detection of a part of sgrnas in example 5 of the present invention, where 1 to 10 indicate sgrnas 1 to 10, Con indicates a sample that was not treated with the sgrnas, and M indicates a DNA marker.

Fig. 5B shows the results of detecting the activity of another sgRNA in example 5 of the present invention, where 11 to 20 indicate sgrnas 11 to 20, Con indicates a sample that was not treated with sgRNA, and M indicates a DNA marker.

Fig. 6 is a diagram showing the electrophoresis results of sgRNA in vitro transcription products in example 6 of the present invention.

FIG. 7 is a diagram showing the results of PCR identification of F0 generation mice in example 8 of the present invention. Panel A shows the result of PCR identification of the 5' homology arm of mouse generation F0; b is the PCR identification result of the 3' homology arm of the F0 mouse; numbers indicate the mouse numbers of F0 generations; m is 1kb DNA marker.

FIG. 8 is a diagram showing the results of PCR identification of F1 generation mice in example 8 of the present invention. Panel A shows the result of PCR identification of the 5' homology arm of mouse generation F1; b is the PCR identification result of the 3' homology arm of the F1 mouse; numbers indicate the mouse numbers of F0 generations; m is 1kb DNA marker.

FIG. 9 shows the results of verifying the expression of murine and human CD40 proteins in wild-type and humanized mice at the RNA level in example 9 of the present invention.

FIG. 10 is a flow cytometry assay of CD40 protein expression in peripheral blood B cells of wild type mice and CD40 humanized homozygote mice according to example 10 of the present invention. hCD40 is a flow antibody against human CD 40; mCD40 is a flow antibody against murine CD 40. Wild type: a wild-type mouse; homozygous: humanized homozygous mice.

FIG. 11 shows the results of flow cytometry for the expression of murine and human CD40 in B cells, T cells, macrophages and dendritic cells of humanized homozygote mouse bone marrow of CD40 in example 10 of the present invention. HCD40 is a flow antibody against human CD 40; mCD40 is a flow antibody against murine CD 40.

FIG. 12 shows the results of tumor volume inhibition and mouse body weight change against the antitumor effect of the human CD40 antibody in example 11 of the present invention.

FIG. 13 is the tumor weight inhibition result and the tumor photograph showing the antitumor effect against the human-derived CD40 antibody in example 11 of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to specific examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The mouse strain, biochemical reagent and experimental instrument used in the embodiment of the invention comprise:

primer synthesis and sequencing services were purchased from bio-engineering (shanghai) gmbh;

In-Fusion HD Cloning Kits available from Takara under the cat number 639650;

XbaI restriction enzyme was purchased from NEB under accession number R0145V;

the sgRNA in vitro transcription kit was purchased from Ambion under the cat number AM 1354;

coli DH 5. alpha. competent cells were purchased from TaKaRa, cat # 9057;

cas9mRNA source Sigmaaldrich, with the product number CAS9MRNA-1 EA;

c57BL/6, ICR mice were purchased from Shanghai Ling Biotech, Inc.;

t7 endonuclease I detection kit, purchased from NEB company, with the product number M0302S;

the PCR product Gel recovery Kit was QIAquick Gel Extraction Kit, purchased from QIAGEN, cat # 28706.

Flow antibody information is as follows:

name of antibody	Goods number	Suppliers of goods
			FITC anti-mouse CD45	103108	Biolegend
PE/Cyanine7 anti-mouse CD11b	101216	Biolegend
			BV421 anti-mouse F4/80	565411	BD Biosciences
BV711 anti-human CD40	563397	BD Biosciences
			APC anti-mouse CD40	130-110-547	Miltenyi Biotec

Example 1 sequence design

The mouse Cd40 gene and the human CD40 gene both contain multiple transcripts, and the sequence design of this example is mainly illustrated by taking one of the transcripts as an example. That is, the 2 nd to 5 th exons (based on the transcript with NCBI accession number NM-011611.2 → NP-035741.2, the mRNA sequence is shown as SEQ ID NO: 1, and the corresponding protein sequence is shown as SEQ ID NO: 2) of mouse Cd40 Gene (NCBI accession number NM-011611.2 → NP-035741.2) are replaced by the 2 nd to 5 th exons (based on the transcript with NCBI accession number NM-001250.6 → NP-001241.1, the mRNA sequence is shown as SEQ ID NO: 3, and the corresponding protein sequence is shown as SEQ ID NO: 4) of human CD40 Gene (Gene ID: 958), wherein the comparison of mouse Cd40 with human CD40 Gene is shown in schematic diagram 1, the finally obtained schematic diagram of the modified humanized mouse CD40 Gene is shown in FIG. 2, and the genomic DNA sequence (CD 40 Gene DNA) of the humanized mouse Cd40 Gene is shown as SEQ ID NO: and 5, as follows:

SEQ ID NO: 5 lists the DNA sequences related to the modified parts, wherein the italic underlined region is the inserted human CD40 gene sequence, the normal italic is a partial sequence of intron 5 of the mouse Cd40 gene, the bold italic is the other sequence introduced for introducing the human CD40 gene sequence, and the normal sequences at both sides are the unmodified mouse genome sequence.

The CDS region and mRNA sequence of the humanized mouse CD40 after being transformed and the protein sequence coded by the CDS region and mRNA sequence are respectively shown as SEQ ID NO: 6. SEQ ID NO: 7 and SEQ ID NO: shown in fig. 8.

Since there are multiple transcripts in the human CD40 gene and the mouse CD40 gene, the method of humanized sequence design in this example is also applicable to the humanized modification of other transcripts. The transcript of the mouse Cd40 gene described above may be replaced with another transcript.

Example 2 design and construction of recombinant vector PBR322-CD40

Based on the sequence design, the inventors further designed the targeting protocol as shown in FIG. 3 and a vector comprising a 5 'homology arm, a human CD40 gene fragment, and a 3' homology arm. Wherein the 5 'homology arm (SEQ ID NO: 9) is the 164899679-164904205 th nucleotide of NCBI accession No. NC-000068.8, the 3' homology arm (SEQ ID NO: 10) is the 164905758-164909778 th nucleotide of NCBI accession No. NC-000068.8, and the human CD40(SEQ ID NO: 11) gene fragment is the 46121820-46123219 th nucleotide of NCBI accession No. NC-000020.11.

The construction process of the vector is as follows: designing an upstream primer for amplifying 3 homologous recombination fragments (LA, KI and RA) and a downstream primer matched with the upstream primer and the downstream primer and related sequences. Wherein, the 5 'homologous arm corresponds to LA fragment, the insertion sequences such as human CD40 gene fragment and the like correspond to KI fragment, the 3' homologous arm corresponds to RA fragment, and the primer sequences are as follows:

LA(4542bp)：

F：5’-CGCGGTCGACAAGCTaagaacaaagtctgcccttg-3’(SEQ ID NO：12)；

R：5’-ctaaaaacagaagtggacag-3’(SEQ ID NO：13)。

KI(1710bp)：

F：5’-cacttctgtttttagGTCCATCCAGAACCACCCAC-3’(SEQ ID NO：14)；

R：5’-ctcttgcttactgtcccttctcgagtaaagcaactgtctcgcttctgagtacgagtgtcatg-3’(SEQ ID NO：15)。

RA(4051bp)：

F：5’-gacagtaagcaagagtatgtgtatgtgtgcatgtg-3’(SEQ ID NO：16)；

R：5’-CGACTCTAGAGGATCccccagctatcttcttactt-3’(SEQ ID NO：17)。

and (2) carrying out PCR amplification by taking C57BL/6 mouse genome DNA or a BAC library as a template to obtain LA and RA fragments, and carrying out PCR amplification by taking knock-in fragments of human CD40 gene fragments and the like of the whole genes as templates to obtain KI fragments. The fragment was ligated to the PBR322-MCS plasmid by means of an In-fusion kit, and the vector PBR322-CD40 was finally obtained.

EXAMPLE 3 verification of the vector PBR322-CD40

Randomly selecting 5 PBR322-CD40 clones, performing enzyme digestion verification by using a restriction enzyme XbaI, and performing electrophoresis on enzyme digestion products to generate fragments with the sizes of 6.3kb, 4.2kb, 3.3kb and 2.3 kb. The cleavage results are shown in FIG. 4.

The plasmid digestion results are all in line with expectations, which indicates that the plasmid digestion verification result is correct. The plasmid was verified to be correct by sequencing company for subsequent experiments.

Example 4 design of sgRNA of CD40 Gene

The sgRNA target sequence determines its targeting specificity and efficiency of inducing Cas9 to cleave the gene of interest. Therefore, efficient and specific target sequence selection and design are a prerequisite for constructing sgRNA expression vectors.

sgRNA sequences that recognize the 5 'target site (sgRNA1-sgRNA10), the 3' target site (sgRNA11-sgRNA20) were designed and synthesized according to the targeting protocol.

Taking a mouse as an example, according to the function and sequence characteristics of the CD40 gene, a 5 'end target site is positioned in intron 1 of the mouse CD40 gene, a 3' end target site is positioned in intron 5 of the mouse CD40 gene, and the target site sequence of each sgRNA on the CD40 gene is as follows:

sgRNA-1 target site sequence (SEQ ID NO: 18): 5'-acctctccctaaccaatgacagg-3', respectively;

sgRNA-2 target site sequence (SEQ ID NO: 19): 5'-cctctccctaaccaatgacaggg-3', respectively;

sgRNA-3 target site sequence (SEQ ID NO: 20): 5'-cctgactttccccacccccatgg-3', respectively;

sgRNA-4 target site sequence (SEQ ID NO: 21): 5'-gactttccccacccccatggtgg-3', respectively;

sgRNA-5 target site sequence (SEQ ID NO: 22): 5'-ctcccaaagacgcagcaggtagg-3', respectively;

sgRNA-6 target site sequence (SEQ ID NO: 23): 5'-ggtgaatgtgaccttgtggctgg-3', respectively;

sgRNA-7 target site sequence (SEQ ID NO: 24): 5'-accttgtggctggcttaagg-3', respectively;

sgRNA-8 target site sequence (SEQ ID NO: 25): 5'-tgaccttgtggctggcttaaggg-3', respectively;

sgRNA-9 target site sequence (SEQ ID NO: 26): 5'-cttagttaatagaagcctgttgg-3', respectively;

sgRNA-10 target site sequence (SEQ ID NO: 27): 5'-cccttttcagttcactttcttgg-3', respectively;

sgRNA-11 target site sequence (SEQ ID NO: 28): 5'-ttttcagttcactttcttggtgg-3', respectively;

sgRNA-12 target site sequence (SEQ ID NO: 29): 5'-tttcagttcactttcttggtggg-3', respectively;

sgRNA-13 target site sequence (SEQ ID NO: 30): 5'-ctccctacctgctgcgtctttgg-3', respectively;

sgRNA-14 target site sequence (SEQ ID NO: 31): 5'-ccaagaaagtgaactgaaaaggg-3', respectively;

sgRNA-15 target site sequence (SEQ ID NO: 32): 5'-gtgcatctgttcggattagaggg-3', respectively;

sgRNA-16 target site sequence (SEQ ID NO: 33): 5'-ctttaagaagagacttcaagagg-3', respectively;

sgRNA-17 target site sequence (SEQ ID NO: 34): 5'-gaagagacttcaagaggatatgg-3', respectively;

sgRNA-18 target site sequence (SEQ ID NO: 35): 5'-aagaggatatggagaccaacagg-3', respectively;

sgRNA-19 target site sequence (SEQ ID NO: 36): 5'-tgtcccttaagccagccacaagg-3', respectively;

sgRNA-20 target site sequence (SEQ ID NO: 37): 5'-caccagtaagtgacccaccatgg-3' are provided.

Example 5 sgRNA screening for different target sites

The activity of multiple sgrnas was detected using a kit. The detection result is shown in fig. 5A and 5B by using a T7 endonuclease I detection kit, and the sgrnas have different activities, so that the sgRNA-5 and the sgRNA-19 are preferentially selected as the sgrnas of subsequent experiments.

Example 6sgRNA in vitro transcription

Synthesis of sgRNA template was amplified by in vitro PCR, and HiScribe was used as a substrate^TMT7 High Yield RNA Synthesis Kit for in vitro transcription.

PCR primers of in vitro transcription template DNAs of the sgRNA-5 and the sgRNA-19 are respectively as follows:

SgRNA-F4(SEQ ID NO：38)：5’-gcTAATACGACTCACTATAgctcccaaagacgcagcaggtGTTTTAGAGCTAGAAATAGCAAG-3’；

SgRNA-F18(SEQ ID NO：39)：5’-gcTAATACGACTCACTATAgccaagaaagtgaactgaaaaGTTTTAGAGCTAGAAATAGCAAG-3’。

the downstream PCR universal primer is:

SgRNA-R(SEQ ID NO：40)：5’-aaaAGCACCGACTCGGTGCC-3’。

a fragment DNA containing sgRNA scaffold was synthesized by DNA synthesis, with sequence information:

sgRNA scaffold(SEQ ID NO：41)：

5’-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT-3’。

products are respectively amplified by using fragment DNA containing sgRNA scaffold as a template and four primers (SgRNA-F4, SgRNA-R, SgRNA-F18 and SgRNA-R), and PCR products are recovered as a transcription template by using a gel recovery kit. The sgRNA-5 and sgRNA-19 were transcribed in vitro as described in the protocol of the in vitro transcription kit. The results of in vitro transcript electrophoresis are shown in FIG. 6.

Example 7 fertilized egg microinjection and embryo transfer

Taking fertilized eggs of a C57BL/6 mouse, and injecting pre-mixed sgRNA-5, sgRNA-19 (transcribed by using an Ambion in vitro transcription kit according to a method provided by an instruction), Cas9mRNA and PBR322-CD40 plasmids into cytoplasm or nucleus of the fertilized eggs of the mouse by using a microinjector. Microinjection of embryos is performed according to the method in the manual for mouse embryo manipulation experiments (third edition), fertilized eggs after injection are transferred to a culture solution for short-term culture, and then are transplanted to the oviduct of a recipient ICR female mouse to produce a genetically modified humanized mouse, so that a first established mouse (i.e., a launcher mouse, generation F0) is obtained. The obtained F0 mouse is crossed with wild mouse to expand population number, and then is self-crossed to obtain homozygote mouse, and a stable hCD40 humanized mouse strain is established.

Example 8 identification of humanized mice of CD40

1. Genotyping of homologous recombination positive mice of generation F0:

the mouse tail genomic DNA of F0 hCD40 mice obtained by using two pairs of primer pairs is respectively subjected to PCR identification, wherein a primer I is positioned on the left side of a 5 'homology arm, a primer IV is positioned on the right side of a 3' homology arm, and a primer II and a primer III are positioned on an inserted fragment, and the specific sequences are as follows:

5' homologous arm recombination identification primer:

primer I: 5'-TTCCTTTCGCAGAGGTGGTCT-3' (SEQ ID NO: 42);

and (3) primer II: 5'-CTCTTGCTTACTGTCCCTTCTCG-3' (SEQ ID NO: 43).

The PCR reaction (20. mu.L) is shown in Table 1:

TABLE 1 PCR reaction System (20. mu.L)

Reaction components	Volume (μ l)
		ddH2O	11.4
5×PrimeStar GXL PCR Buffer	4
		2.5mM dNTP	2
Primer I(10pmol/μl)	0.4
		Primer II(10pmol/μl)	0.4
PrimeStar GXL DNA Polymerase*	0.8
		Genomic DNA	1
Total of	20

The PCR amplification reaction conditions are shown in Table 2:

TABLE 2 PCR amplification reaction conditions

Step # of	Temperature (. degree.C.)	Time	Remarks for note
				1	94	3min	-
2	98	15sec	-
				3	64	15sec	-
4	68	2min	Repeating the steps for 2-435 cycles
				5	68	5min	-
6	12	-	Heat preservation

3' homologous arm recombination identification primer:

and (3) primer III: 5'-CAGAAGCGAGACAGTTGCTTTACT-3' (SEQ ID NO: 44);

and (3) primer IV: 5'-TTACTTTGTGGCACTGGTCTAACC-3' (SEQ ID NO: 45).

The PCR reaction (20. mu.L) is shown in Table 3:

TABLE 3 PCR reaction System (20. mu.L)

Reaction components	Volume (μ l)
		ddH2O	11.4
5×PrimeStar GXL PCR Buffer	4
		2.5mM dNTP	2
Primer III(10pmol/μl)	0.4
		Primer IV(10pmol/μl)	0.4
PrimeStar GXL DNA Polymerase*	0.8
		Genomic DNA	1
Total of	20

The PCR amplification reaction conditions are shown in Table 4:

TABLE 4 PCR amplification reaction conditions

If the insertion position of the recombinant vector is correct, only 1 PCR strip with the length of 6.5kb is needed for identifying the primer pair I and II by the 5 'homology arm, and 1 PCR strip with the length of 4.3kb is needed for identifying the primer pair III and IV by the 3' homology arm; negative mice should have no bands.

The PCR identification of F0 mouse is shown in FIG. 7, in which 3 mice numbered 5, 7, 9 are positive.

2. Genotype identification of F1 mouse:

f1 generation mice were obtained by mating F0 generation positive mice with wild type mice. PCR identification was performed on F1 mouse tail genomic DNA. PCR conditions and primers were identified with the genotype of F0 mouse. The results of the F1 mouse PCR experiment are shown in FIG. 8, which shows that 11F 1 mice are positive, specifically numbered 2, 3, 6, 7, 10, 11, 15, 16, 19, 20, 23.

The obtained F1 mouse generation was mated with a wild type mouse, respectively, to propagate the population. Meanwhile, the heterozygote mice are selfed to obtain homozygote mice.

Example 9 humanized mouse CD40 RNA expression analysis

Two wild-type mice and one hCD40 homozygote mouse (6 weeks old) are selected to extract total spleen and thymus RNA, reverse transcription is carried out to cDNA by using a reverse transcription kit, and PCR is carried out, wherein the two contents are mainly verified: 1) verifying whether the humanized transformation of the human fragment insertion influences the normal shearing of the CD40 gene; 2) and (3) verifying whether the humanized insert is expressed in the humanized mouse.

Aiming at the experimental purpose, 3 pairs of PCR primers are respectively designed, the general positions of the primers are shown in FIG. 9 and A, the P1/P2 primer pair and the P3/P4 primer pair are on humanized CD40 mRNA, the primers P1 and P4 are on non-humanized exons, the primers P2 and P3 are on humanized exons, the P1/P2 primer pair can judge whether the exon 1 and the exon 2 are correctly sheared after humanized modification, the P3/P4 primer pair can verify whether the exon 5 and the exon 6 are correctly sheared after humanized modification, and the primer pairs P1/P2 and P3/P4 can reflect whether the humanized CD40 sequence in a humanized mouse is expressed. The primer pair P5/P6 is located in the corresponding humanized replaced region of the mouse Cd40 gene, and the primer pair can be used for identifying whether murine non-humanized mRNA is expressed in a CD40 humanized homozygote mouse.

The primer pair P1/P2 has the sequence:

P1：5’-CCCTGTGATTTGGCTCTTCT-3’(SEQ ID NO：46)；

P2：5’-GGTGTCTAGGAATTCGCTTTCA-3’(SEQ ID NO：47)。

the primer pair P3/P4 has the sequence:

P3：5’-GAAGGCTGGCACTGTACG-3’(SEQ ID NO：48)；

P4：5’-CATTAGTCTGACTCGTTCCTTTCT-3’(SEQ ID NO：49)。

the primer pair P5/P6 has the sequence:

P5：5’-ACAAACAGTACCTCCACGATG-3’(SEQ ID NO：50)；

P6：5’-TTCTTAACCCGAAGCCCTTG-3’(SEQ ID NO：51)。

the PCR reaction conditions of the primer pairs P1/P2, P3/P4 and P5/P6 are as follows:

the PCR reaction system is 20 mu L, and the reaction conditions are as follows: 95 ℃ for 5 min; (95 ℃, 30 sec; 60 ℃, 30 sec; 72 ℃, 55sec, 35 cycles); 72 ℃ for 5 min; keeping the temperature at 12 ℃.

The results of PCR reactions with primer pairs P1/P2, P3/P4, P5/P6 are shown in FIG. 9, B: the P1/P2 primer pair and the P3/P4 primer pair have no PCR products amplified in a wild mouse sample, and PCR products of 244bp and 232bp can be amplified in a humanized homozygote mouse sample respectively, which indicates that the expression of the humanized CD40 mRNA can not be detected in the wild mouse, and the active expression of the humanized CD40 mRNA can be detected in the humanized homozygote mouse; the P5/P6 primer pair amplified a 196bp PCR product in the wild type mouse sample, while no PCR product was amplified in the humanized homozygote mouse, indicating that the humanization strategy was successful in the humanized homozygote mouse with no detectable expression of murine RNA for the humanized region.

Example 10 humanized mouse CD40 protein level expression assay

Normally, CD40 is expressed in B cells, macrophages, dendritic cells, vascular endothelial cells, etc., and in order to verify the expression of humanized mouse CD40 protein, we examined the expression of murine and human CD40 on peripheral blood B cells of wild type mice and humanized homozygote mice, and the expression of murine and human CD40 on bone marrow B cells, T cells, macrophages and dendritic cells of humanized homozygote mice, respectively, the brief procedure and results are as follows:

1) the heterozygous mice are selfed to obtain CD40 humanized homozygote mice and wild type mice, one hCD40 homozygote mouse and one littermate wild type mouse (6-7 weeks old) are selected, peripheral blood is taken from the inner canthus, and after red blood cells are lysed, antibody is added for incubation, and flow cytometry detection is carried out. After live cells were selected by flow cytometry, B cells were sorted out using antibodies against murine CD45 and CD19, and then expression of CD40 protein on B cells was detected using antibodies against murine CD40(mCD40) and human CD40(hCD40), respectively, as shown in fig. 10, showing: the expression of murine Cd40 protein can be detected only on wild type mouse B cells, and the expression of human CD40 protein can not be detected; on B cells of a CD40 humanized homozygote mouse, only the expression of human-derived CD40 protein can be detected, and the expression of murine Cd40 protein cannot be detected.

2) The heterozygote mouse is selfed to obtain a CD40 humanized homozygote mouse, bone marrow cells of the homozygote mouse are taken, red blood cells are lysed, antibodies are added for incubation, and flow cytometry detection is carried out. After live cells were selected by flow cytometry, CD45 positive cells were selected by antibody against murine CD45, and then B cells and T cells were selected by antibody against CD19 and antibody against CD3, respectively; (ii) in cells negative for CD3/CD19, macrophages were selected using antibodies directed against F4/80 and antibodies directed against CD11 b; dendritic cells were selected using antibody circles against CD11c and against IA-IE. In the above cell types, the expression of CD40 protein was detected with antibodies against murine and human CD40, respectively, and as a result, as shown in fig. 11, it was found that, in the normal case, active expression of human CD40 protein was detected on B cells, macrophages and dendritic cells of humanized homozygous mice, no expression of murine CD40 protein was detected, and no expression of CD40 protein was detected on T cells.

Example 11 in vivo validation of drug efficacy of a humanized animal model of CD40

CD40 humanized homozygote mice and wild type mice were obtained by mating heterozygote mice and subjected to in vivo efficacy verification experiments, the experimental groups and dosing are shown in the following table:

6-8 weeks old CD40 humanized mice and C57BL/6 wild type mice were subcutaneously inoculated with mouse colon cancer cells MC38 (1X 10)⁶/100 μ l pbs), when tumors grew to a volume of about 100 cubic millimeters, the CD40 humanized mice were randomly assigned to groups 1, 3, 4, 5. Group of1 is a control group; groups 2, 3, 4, and 5 were administration groups, to which antibody drugs (selecrelumab analogues) against the human-derived CD40 target were administered, respectively. Tumor volume and mouse body weight were measured 2 times a week on days three and seven respectively, and the experiment was terminated at 3 weeks.

The tumor growth inhibition results using tumor volume as an indicator are shown in fig. 12, a: after 6 doses, by the end of the 21-day experiment, no significant tumor suppression effect was seen in the group 2C57BL/6 wild-type mice with the antibody drug against the human CD40 target at the same dose (10mg/kg) relative to group 1 (control-saline group): tumor inhibition (TGI) 7.98%; whereas a very significant tumor suppression effect was observed in the CD40 humanized homozygote mouse: the tumor inhibition ratio (TGI) was 86.23%, and the differential significance P value was 0.00135; humanized mouse administration group: groups 3, 4 and 5 showed significant tumor suppression effects on tumors; meanwhile, the antibody drug aiming at the human CD40 target point shows a certain dosage effect among groups 3, 4 and 5, and the tumor inhibition effect of the groups 4 and 5 (two low-dosage administration groups) is obviously lower than that of the group 3 (high-dosage administration group). The results of the body weight measurements (fig. 12, B) for each group during the administration were similar to the results of tumor growth inhibition using tumor volume as an indicator, with no significant difference between group 2 and group 1, and group 3 exhibited significant weight inhibition relative to group 1; the body weight measurements between groups 3, 4, 5 showed some dose effect, with groups 4 and 5 (two low dose groups) having significantly less effect on body weight than group 3 (high dose group).

To further confirm the tumor growth inhibition results using tumor volume as an index, tumors of each tumor-bearing group of mice were dissected on the last day of the experiment, and the tumor weights of each group of mice were compared, and the data are shown in the following table, and the analysis results are shown in fig. 13, a.

The comparison of tumor weight and tumor volume size for each group of mice was similar: the antibody drug against the human CD40 target did not show significant tumor suppression effect on the C57BL/6 wild-type mice of group 2 at the same dose (10mg/kg) compared to group 1 (control-saline group): the tumor inhibition rate is 11.70%, the P value is 0.572, and no significant difference exists; whereas a very significant tumor suppression effect was observed in the CD40 humanized homozygote mouse: the tumor inhibition rate is 85.48%, and the differential significance P value is 0.00173; groups 3, 4 and 5 showed significant tumor suppression effects on tumors; meanwhile, the antibody drug aiming at the human CD40 target point shows a certain dosage effect among groups 3, 4 and 5, and the tumor inhibition effect of the groups 4 and 5 (two low-dosage administration groups) is obviously lower than that of the group 3 (high-dosage administration group).

The above results show that, after humanization of the CD40 gene in mice, it can be recognized by an antibody drug (selibrelumab analog) against a human-derived CD40 target, and shows significant anti-tumor effects at both high, medium and low doses, and shows dose dependence; on the other hand, in wild-type mice, the murine CD40 target was not recognized by the Sericilumab analog, and thus had no antitumor effect. Therefore, the humanized mouse model of CD40 can be used as an anti-tumor efficacy evaluation model of antibody drugs aiming at the human CD40 target.

The embodiment of the invention provides a method for preparing a CD40 humanized mouse model and verifies the application of the mouse model. The humanized mouse of CD40 established by the method provides a transformable model, which has a humanized CD40 receptor and a functional mouse immune system, and has the following advantages: 1) can be used for testing the drug effect of an agonistic antibody or other molecules specific to the human CD40 target in vivo; 2) the toxicity and safety of the drug against the CD40 target can be assessed in vivo; 3) compared with non-human primate models and the like, the model provides a more economical and efficient in-vivo research model selection.

Sequence listing

<110> Shanghai's Square model Biotech Co., Ltd

Shanghai Yushi Biological Technology Co., Ltd.

Guangdong Nanmo Biological Technology Co., Ltd.

Construction method and application of humanized CD40 gene modified animal model

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His Asp Gly Gln Cys Cys Asp Leu Cys Gln Pro Gly Ser Arg Leu Thr

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Arg His Cys Glu Pro Asn Gln Gly Leu Arg Val Lys Lys Glu Gly Thr

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Ala Glu Ser Asp Thr Val Cys Thr Cys Lys Glu Gly Gln His Cys Thr

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Ser Lys Asp Cys Glu Ala Cys Ala Gln His Thr Pro Cys Ile Pro Gly

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Phe Gly Val Met Glu Met Ala Thr Glu Thr Thr Asp Thr Val Cys His

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His Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile

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Ser Val Gln Glu Arg Gln Val Thr Asp Ser Ile Ala Leu Arg Pro Leu

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gaagattatc ccggtcataa caccgctgct ccagtgcagg agacgctgca cgggtgtcag 780

cctgtcacac aggaggatgg taaagagagt cgcatctcag tgcaggagcg gcaggtgaca 840

gacagcatag ccttgaggcc cctggtctga 898

<210> 7

<211> 2986

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gggacuccua gcagggacuu uggagugacu uguggcuuca gcaggagccc ugugauuugg 60

cucuucugau cucgcccugc gauggugucu uugccucggc ugugcgcgcu auggggcugc 120

uuguugacag cgguccaucc agaaccaccc acugcaugca gagaaaaaca guaccuaaua 180

aacagucagu gcuguucuuu gugccagcca ggacagaaac uggugaguga cugcacagag 240

uucacugaaa cggaaugccu uccuugcggu gaaagcgaau uccuagacac cuggaacaga 300

gagacacacu gccaccagca caaauacugc gaccccaacc uagggcuucg gguccagcag 360

aagggcaccu cagaaacaga caccaucugc accugugaag aaggcuggca cuguacgagu 420

gaggccugug agagcugugu ccugcaccgc ucaugcucgc ccggcuuugg ggucaagcag 480

auugcuacag ggguuucuga uaccaucugc gagcccugcc cagucggcuu cuucuccaau 540

gugucaucug cuuucgaaaa augucacccu uggacaagcu gugaggauaa gaacuuggag 600

guccuacaga aaggaacgag ucagacuaau gucaucugug guuuaaaguc ccggaugcga 660

gcccugcugg ucauuccugu cgugaugggc auccucauca ccauuuucgg gguguuucuc 720

uauaucaaaa agguggucaa gaaaccaaag gauaaugaga ucuuaccccc ugcggcucga 780

cggcaagauc cccaggagau ggaagauuau cccggucaua acaccgcugc uccagugcag 840

gagacgcugc acggguguca gccugucaca caggaggaug guaaagagag ucgcaucuca 900

gugcaggagc ggcaggugac agacagcaua gccuugaggc cccuggucug aacccuggaa 960

cugcuuugga ggcgauggcu cggcucggga gcaggggccu ggcucugagg acugcuugcu 1020

gaccuuugaa guuugagaug agccaagaca gagcccagug cagcuaacuc ucaugccugc 1080

ccccuaucau uucucaacuu gcuuuuuaag gauggaggga gagcucgggc aucggggguc 1140

cacagugaua ccuaccaagu gcagcagugc aggacccaga gucgucuugc ugcggcguuc 1200

acuguaagga gucauggaca caggaguccg uggcccacag cuugugcugc uagagggcac 1260

cugguugccc aucagcaggg uacuggcuaa auaaaucugu aauuauuuau acaaugacau 1320

cucagaaacu cuagcaggug gggcagaaaa cagguaguag aaugaugggu agagaaauag 1380

cuuuuaaaac acauuccaag gcagguaaga uggcuuuugu gaguaaagga gcuugcugcc 1440

caaacccggu uaccugauuu ugaucccugg gacuucaugg uaaaagggag agaaccaaau 1500

ccagaggguu gucauuugac cuccaugugu gcucuguggu aauguacccc gugugugcac 1560

augugcacau auccuaaaau ggauguggug guguauugua gaaauuauuu aaucccgccc 1620

ugggguuucu accugugugu uaccauuuag uucuugaaua aaagacacac ucaaccuuua 1680

uauuuacaau aagccuuaaa cagcacaaga acugggcacc ugccuacccu guaugcuauu 1740

ugaaucuacu uuccuauuga uaacccccag uuauuacuua cuacuuacua uguuucaucg 1800

gggcugcucu uaucuccuau uggccagccc cagguccaug uucucuuaaa cucuccuaau 1860

ccaugguggc uucucuuucc uccauucacc ucuucucucu cuugguucuc uucuaacgcc 1920

aagcccagga auacuaagcc ccaccuaugu cucuucuguc uagcuauugg cugcuggcau 1980

cuuuauuuac caaucagaaa uaacuugggg ucacuugggg ggguugucua caguacagac 2040

uccaggucuu ggggccacaa acccauuaca auacagagca agaucaaacc ucaacagugg 2100

ugcaugccug uaacccuagc acucaggagg aagaggacua ggaguucaag gcuagccuug 2160

gauauauagu uaacucaagg ucagccuggg cuacaugauu gucuuaguug ggguuuuauu 2220

gcuguaaaca gacaccauga gcaaggcaac ucuuaaaagg acaacauuua auuggggcug 2280

gcuuacaggu ucagagguuc aguccauuau ccucaaggca ggaacauggc agcauccagg 2340

caggcauggu gcaggaggac cugagaguuc uauaucuuca ucugaaggcu gcuaggcuga 2400

agcugacuuc cagacagcua gggugagggu cuuaaagccc acacccacag ugacacaccu 2460

acuccaacaa ggccacacau ccuaauagug ccacucccug ggccuagcau uuacaaacca 2520

ucacaaugau acugucucaa aaacaaacau uaaagagcaa caacuucucc caacaacccg 2580

aaccauacac acaaagccac acauccuuca aacaguuuuu auauacgaua ugacaguaug 2640

uaagcaggcu gcauauuaua uuugaauaca guauguauag auauggaagg aaagcuaaga 2700

uaccaugaau gccugugugg guaaaacaaa gcauugaaaa ucccaacuuu gacagugguc 2760

uagcgagaug ggucagggcu aggagcagca gcuccugcga aggcucuggg uuuaguuucc 2820

agcccucaca cagggcauau guguaucaug ugugcacugu ccacacaggu ggaacacuug 2880

uacacauaau auaaacgaac uaucuuuuuu aaaaaaagaa uggugugguc ugaagugggu 2940

gggugggagu guuguguuuc caauaaugcu ucauuauuuu uccaag 3084

<210> 8

<211> 289

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Val Ser Leu Pro Arg Leu Cys Ala Leu Trp Gly Cys Leu Leu Thr

1 5 10 15

Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu

20 25 30

Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val

35 40 45

Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu

50 55 60

Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His

65 70 75 80

Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr

85 90 95

Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr

100 105 110

Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly

115 120 125

Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu

130 135 140

Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys

145 150 155 160

Cys His Pro Trp Thr Ser Cys Glu Asp Lys Asn Leu Glu Val Leu Gln

165 170 175

Lys Gly Thr Ser Gln Thr Asn Val Ile Cys Gly Leu Lys Ser Arg Met

180 185 190

Arg Ala Leu Leu Val Ile Pro Val Val Met Gly Ile Leu Ile Thr Ile

195 200 205

Phe Gly Val Phe Leu Tyr Ile Lys Lys Val Val Lys Lys Pro Lys Asp

210 215 220

Asn Glu Ile Leu Pro Pro Ala Ala Arg Arg Gln Asp Pro Gln Glu Met

225 230 235 240

Glu Asp Tyr Pro Gly His Asn Thr Ala Ala Pro Val Gln Glu Thr Leu

245 250 255

His Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile

260 265 270

Ser Val Gln Glu Arg Gln Val Thr Asp Ser Ile Ala Leu Arg Pro Leu

275 280 285

Val

<210> 9

<211> 4527

<212> DNA

<213> Mouse (Mouse)

<400> 9

aagaacaaag tctgcccttg taagccccaa tctgggctcc agggaggggc tgggccctga 60

gagaaatagc aggtgttctt tactgacctc ttcctgtgca gggggctgtg agggagactg 120

catgtcacct tcctgtgtga gggagactgc atgtcacctt catttgatgt ctcccaacgt 180

ctctgttaca cggatgacca agatgaggct cagaaagggg aggggtttta cccaaagcct 240

cttgacatgg atatgaagct atgtctgcct gacttctgag cctgccactc acgcggcaag 300

gggtcttcaa gctgtggccc tgagacctat tgtggaggaa ggaacaatgc tctgggggct 360

ggaaactggg gctgactggc catcagaggg aaagtgtctt ttccaaaggt gccattgagg 420

atgtctactg ttctagaagg aaggaaggaa ggaaggaagg aaggaaggaa ggaaggaagg 480

aaggaaagga gggagggagg gagggaggtg ggaagtctca gggaagataa aactcaggga 540

tgatgccctg ctgggggcag gtgctttcag gtacatttca ttaagtgatt tgctcagata 600

cccagtttga gtgagagttc tcaatttcct ggattctgga cagtcagaca gctactaggg 660

tgcaggacac cagggcagac ccccatcatc tatgtgtgct gattaaagct gccattttct 720

gaatactacc ttgctatact gccaggccat gggcaagtgt tacaatctca ttttaaaggt 780

gagaacacag ggctagagag atggcttagt gggtaagagc gcacgctggc tgccatagcc 840

acattgggct ggtacataaa ttcaacaaga ataataaaaa caaaggaaac acagtcttag 900

agaagcaaaa taattaggat ggtgtactgg ctagttttgt gtgtcaactt gacacaggct 960

ggagttatca caaagaaagg ggcttcagtt ggggaaatgc ctccatgaga tccagctgta 1020

aggcattttc tcaaatagtg atcaagtggg gaggtcccct tgtgggtggt gccatctctg 1080

ggctggtagt cttggttcta taagagagca ggctgagcaa gccaggggag gcaagccagt 1140

aaagaacatc cctccatggc ctctgcatca gctcctgatt cctgacctgc ttgaattcca 1200

gtcctggctt ccttagtgat gaacagcagc atgaaagtgt aagctgaata aactctttcc 1260

tccccaagtg cttcttgatc gtgatgtttg tgcaggaata gaagccctga ctacacagat 1320

gggatacaga gtggtttgtc ccgttctcaa atgtgggctc cacgcaatcg gtagctgtgg 1380

ttgtttctaa aatgtaacct ggcttcaagg agcacacgag caagccggag ggcttagctc 1440

tgcagttaag agcatgtgct gcccttgcag agagctggaa tccgattccc agctcccaca 1500

ttccacagcc cccagatatc tgatcccctc ttccgagggt acttacaccc acacatgttc 1560

atattcatat atctacctat aatcaaacat gaaataaatc ttgtggctag agagatgctc 1620

agaatattgt tgctctggga gaggacccag gttcagtttt ctagcaccca cgtgtggccc 1680

acaatcatcc tcaacaccag ttccagggaa cccagtgccc tcttctgacc tccaagggca 1740

ccaggcacgc atgtggagca tattcaggct acacttgtac acataaaata aataaatcta 1800

attattttta aagtcttaaa aaacaaacaa acaaaccccc aaacaccgtc gcctaaaacc 1860

tcaaggatag ggttgtgact gaaggcactt gtcatataag cctgaggccc tgggttcatt 1920

tcctggaacc catgtaaggt agtgtgtggt ggcagaagtc caagtctgac atcctaaggc 1980

tcctactgtg agacaggagg tgggaacagg agaaccccgg aaactttcag cacatgcaac 2040

caagaacaac aaaatgatgg tggcttaagc acagtaggag atagcactga gagcagtgtt 2100

ggccactgac ctccgtacac ttccagttat ggcaagagca cgctccaagc tgcagacagg 2160

gcagtgggcg ttgcgtgtgc acacacacca aaacacgggc ccactttatg gttcttaaaa 2220

aaagtttcta cacttaaaaa aatgtagttt tcgttatact tatttattta tatattattt 2280

gtatgcatgc atgcatgcat gcgtgcttga gtaccaaagc gtgtgtgtgg tcagaggaca 2340

agttagggaa gctggctctt cctaccatgt gattctaagg aactgaactc aggctgtcag 2400

gcttggcagc aagagccttc accagctgag ccatctctcc ggcacctctc tctctctctc 2460

tctctctctc tctctctctc tctctctctc tctctctcct gtttgtattt ttctttttta 2520

aaaatatttt ttcaagtttt tttttttttt aaagatgtat ttattttatg tgagtacact 2580

gtcactgtct tcagacacac cagaagaggg catcagattc cattacagat ggttgtgagc 2640

taccctgtgc ttgctgggat ttgaactcag gacctttggt cagtgttctt aactgctgac 2700

ccatctctcc agccctgttt gtatttttca acacaaggtt tctctgtgta gccctggccg 2760

ttctagacca ggttggcctc gaactcagag atccacctgc ctctgccgcc tgagcgctgg 2820

gattaaaggt gtgcgccacc actgctcagc tgactgtttc tggaagtgtt acagctccgc 2880

tctgagggaa acttttctct gagtgttgta gatgtgagga gaaatcccga aaggcttccc 2940

tgtggaggtg ttgcatctct gaaaggaaaa gcggagtatc acaaagtccc actgctggac 3000

aaacctcaga gagtggctgc ctccgcccag gggcgaagca gcagagagct gagctgcagg 3060

cagcttaggc agttctccag ggtggagtcc ttctgggcag ggattggtga gacttcatgc 3120

tcaaggattg gtgggttcgc atagttcttt tattttttcc caactaggaa gtgggctcag 3180

gcctttcccc cagctagagt ttcactgttc tttaaaaaac atcattttgt ttttatttca 3240

tgtgaattgg tgttttgccc tgcatgtttc tctgtgtgag ggtgtcagat tccctggatc 3300

tggagttaca actggtggtt gtgagttgcc atgtggatgc tgggaactga acttgggtcc 3360

tctgaaagag taaccagtgc tcttagccat tgagccatct ctccagctcc tccaaatcct 3420

tttcttattc atttaaaaaa ttacatgtat gtatgtatgt atgtatgtat gtatgcatgt 3480

ttgtacgcac acacacacac acacacacac acacggagat tagatgctaa cttttgagag 3540

ttggttctct ccttccattt ctgggccttg aaattctggt tatcagtctt ggcagcaagc 3600

gccttgattg gctgagccat ctcgctcctt ggttcttcaa ggagtaagtc tctggcgcta 3660

gctagatcat agttaatgcc tttttttttt cttttttctt tttttgagac agggtttctc 3720

tgtgtagctt tggctgtctt ggaactcact ctgtagacca ggctggactc acagagatct 3780

gcctgcctct gcctctggag tgctgggatt agaggcttat gccaccacag ttggccagtt 3840

aacacctctg aaagacttgc taccaaccca ccccaggctt aaaagtaaaa tcaagagcag 3900

acagagcgaa ggatctcagc aaagaaagct acgcatcgag gcttaataac cctgttatga 3960

atctgttgag tgtattttta gggtttcttt taatttatag gaagtgatac ttgctgacct 4020

cttgatgcag cagtagaaga tttacagtta aaagaagtgt gcttaaatta gcaagaagca 4080

gctcatagca tgggtggtcc ccggatgttg tagaaacaca tgttgagagt cccgcccctg 4140

tggactctgt tcagtgttgc cctctgtggg gtgattctta tctctttggt ggcagggagc 4200

tggggacaga aaccgggaga agggctgagg ccagcttgag ccagcagtct cgggactctg 4260

gaggaagaac tggagttctc cctacctgct gcgtctttgg gagcactgaa gagtcctgtg 4320

catctgttcg gattagaggg ttctgcgttc ttgctttggt agatggcagt aagacgatgt 4380

gacaacagag taaaaaaaaa aatagacctc acactctggg ggctcacttt tctgctttgg 4440

atttccacat cagctacagc ctgcgtcttg gctaactttc aacatgccgg tggaagatcc 4500

cttccagctg tccacttctg tttttag 4527

<210> 10

<211> 4021

<212> DNA

<213> Mouse (Mouse)

<400> 10

tatgtgtatg tgtgcatgtg tatgcatgtg catgcatgca tgtgtgtgtg catgcatgta 60

tgagcatata tatgcatgtg catgcatgga tgcatgtgca cacatgcatg tatgtgcatg 120

tatgtgcatg tgcattcatg catgtgtgca tgcatgtgta tgcatgcatg tgcattcatg 180

catgtgcatt catgcatgtg tgtgcatgtg tcagctatca gtttggtgtg ttcctcctca 240

ggtactgtcc actgtttttt gtttttgttt gtttgtttgt tttgttttgt tttgttttga 300

gagtcccaac actgggacta taaccacttg ctaccaagca tggatttttt tttttttttt 360

atcgtgggtt ccagggcttg aactcgggcc cttgggttca cagtgctttc ctgagtgagt 420

tatcaatgcc cccagccctt ctggactctt acttgtgttt gttgggcttt gtctgggttc 480

tctgaccact tctttccacc tctccttggc tgtcttatct acatctatgg ctccatctcc 540

ccctggatct gtccttcagc ccccagatgg tcaagtccca ctgctgagtg ttatggatca 600

ccagcacctc agactctgtg catccaaaat gggcccctag ccaatccctg agccccagct 660

gacaggcagg ttctggctcc aggcagtcac acagtgagct gcccaccctt gccttcaact 720

ccctgtcagt caggcccata tgactgtctt gggaatccac ctcctcactc tctagagtgg 780

tcacgtgagt tcgcacctcc tcactgtggt gtggtgatgg ctctgtctcc tcataggctc 840

cctgcctctg gtgtctccct tggcagcttt gcaccatgct gctgttggga gcttccgtag 900

ccccttttgg atcatgtcgc ttcatgattt aaagcactcc acgtctccct gctgcctttg 960

ggaggaagcc cagactcccg ggactggggc tgggccttcc agctacagtt cctgtctctc 1020

cccccacacc tcgcaccctg cacttgcacc ccgggcacac tgtttattct tttgcttcaa 1080

agtctcctgt ttgggagaaa acaatatata tgccctattc tgttttttat agttcacatt 1140

taatgttttt taaatgggca aacctgtcac ctcaggctgt ttcctcacta ctccttcaga 1200

gagagagaga cagagagaga cagagacaga gagagacaga gacagagaga gacagagaga 1260

gacagagaca gagactcaga gacagtcaga gagacaaaga tagagacaga gaaagagaca 1320

atcagtgaga gacagaggga tagagagaga cagagagtca gagagacaga gacagataga 1380

gagacagaaa gacagagaca gagaggtggg gggaggagat agggggagac agagggacag 1440

agagacagaa ggacagagaa actgctaata aacaaacaca ttgacatacc actcccctct 1500

gccatgatac acatatgcac acacacacat acacacacac acacacacac acacacacac 1560

acacacacac acacacacac acacacacca cagtggtttc tcttgtggtt atagtgcttg 1620

cttgcaggtc acactcaccg tcctcactgg tgggtttagt tgttcataga agttcccgtc 1680

ccggagtcaa atgtgcaact gccgcactgc cccacttagc tggttcatgc tgctgtttca 1740

acttttatcc cctttggaga cccttcggat cttctctggg gacccccaaa tctgcctcag 1800

tttgtgtgag accctcaggg atgcccctaa tctcgggagg cttcagccaa cttgtaaagg 1860

tgctgagggc ctttctcaca caaggctaga gcacgcacgc attttctctg aagcctctct 1920

tccaccacat cccggttttc cttcgccatg tcccttgcca tgtcccctct gtcccctcct 1980

cccaggacct tccatccaca ccacacatct ccactcctgt cctgcctctg gctgcccact 2040

ctgctgcaac ggtcctctca ccagtcaggt cactccctag ctgcctgcct gcagctgccc 2100

tgcctgcagc tgctcatcac cgcacttgtc accatgtgac tcccctcctg tcacactgtt 2160

cctacttagc tgggtcccac tttcccatcc ttcaatgcct tctgatgccc cccttcatgg 2220

ggaagtcttc ccagaagacc ctgaaagcag agcttcttaa cggggaccag tttggctctc 2280

tgtgggggcg ttggccacat ctgatcatct tttggatgct gtacttggcg gggaggctgt 2340

ttctggcatt tggttagtgg aggcaaaggt gctgctaaat agtctgtgaa acaaaggcca 2400

tttcccagca caaaatacct gcagattggc catttccatt tcaatatgac cgtaaccttt 2460

cctttctgtg gtttctgttc ttcacttaat gatcatcttg ggatgctgca ctctaagtca 2520

cgtgctcagt gaacaaggac ttgctgcttg ctgggggact cccctgggct tggaagtctt 2580

atggcgggga gccctgtttc tgtctgtctg tctgcatgtg tgtgtgtaca tgcacataca 2640

tgtgtacaca tgtgtctttg tgcagctgtg aggataagaa cttggaggtc ctacagaaag 2700

gaacgagtca gactaatgtc atctgtggtg agtccagggg agaatggcct tgccaagtct 2760

ttgggaagca gggaactggg gagagactga ggcacgcagg aacactgact gggataggag 2820

tgagaccaag aggcagtttg gggtacagta ccttagctcc cgtcttggga gctgggtaag 2880

tcacatccct tgtctgagcc tcagtttctt caattgtgaa ataggcccac agcagctcct 2940

tcctccctta cctgggtcgt gtaagtggca ttggaatttt gcagtttgga agctgctgcc 3000

ccttgcttga ggttcaggtt cactgtgaca gtgtcacctg gtaaccccag tttggatgct 3060

aggatgtaaa acttgaccat cccctaatgg atcacaatct cagataacaa tagagaccag 3120

gccacttttg aatgagtgaa gacagagaag ggtaagagag ctaggtctga tgagcgggcc 3180

tgtcagcgca gctaattaga ggcaagagct ttgtaagttc aaggctagcc tgggcagctt 3240

agaaagatac tggttcaaca tagaaaaggg ctgctgagat gggtcagtaa gttaagctct 3300

tgcctgatgg cccagcttcc atccccagca tccatggaaa ggtagaagga gaggatcagc 3360

tcctaaaagt tgtcctctga ccgctgcatg tacagcacaa cacagcacag cacatgtgag 3420

tgtgcataca tcatgcacac acatcataca tacatcatgt gcacatatca tgcacacaca 3480

tcatgtgcac acatcataca cacatcatgc acacacatca tgtgcacaca tcatgcacac 3540

acatcataca tacatcatgt gcacatatta tgcacacaca tcatgtgcac acatcatgca 3600

tacacatcat acacacacat catgcacaca catcatgtgc acatatcata cacacatcac 3660

gtacacacat catgcacaca catcatacac acatatcatg tgcacccatc atgtgcacac 3720

atcatacaca catcatgtac atgcacaccc acaatagtga taaataaaag tttaaatatg 3780

tttctagggc tggggaggtg gcacttgctg ttctgacaga ggacctgggt tcagtttcct 3840

gagcccatgt cattgcagta taaaactgtc catgactcca gctcctggtg atctgatatc 3900

tctgctggcg ctaggcacat acatgatgca cgtacatacc tctagcactt tctgatatac 3960

ataaataaaa atagatacaa attaaaagac attaaaaaaa aaagtaagaa gatagctggg 4020

g 4155

<210> 11

<211> 1400

<212> DNA

<213> Human (Human)

<400> 11

gtccatccag aaccacccac tgcatgcaga gaaaaacagt acctaataaa cagtcagtgc 60

tgttctttgt gccagccagg tgagatgcca accctctagc cccatcatgg agtccccctt 120

tgctttggtg gcagacgcag accccatatg ttaactgtaa actcaaatct gaaacgaccc 180

atttcccagc cctgcttcac tgtcagaatg ttctggttcc ctctctacca ggtaaaactc 240

tgtctaccct gaactaggga tcccagcttc tccatcttcc tcgcctgatt atgaaggatc 300

caagactttc atctttgaat cccctaccct aaagcctggc ctgatcattg tgtggttagt 360

gtctgactca tggagttggc cagagccctc cctcatttcc tgatgttttc caggacagaa 420

actggtgagt gactgcacag agttcactga aacggaatgc cttccttgcg gtgaaagcga 480

attcctagac acctggaaca gagagacaca ctgccaccag cacaaatact gcgaccccag 540

tgcgtgcgct gttgggaaag ggacgcttgg gaaccgggct gatattcccg acaatgcagc 600

cattctaatt ttatgtagcc agggtctgct ctgattggtt ggagtccggg ctgtactgat 660

cattaaatga tttgattgcc atctctactt ggaagagggt ctgaggaaga aagagcaggc 720

aatgtgggga gtgaggctca gagcatggcc cagcaggggg ttcccatcct tcctgccctt 780

ctcttctcag acctagggct tcgggtccag cagaagggca cctcagaaac agacaccatc 840

tgcacctgtg aagaaggctg gcactgtacg agtgaggcct gtgagagctg tgtcctgcac 900

cgctcatgct cgcccggctt tggggtcaag cagattggta agtggctcat ctgggaatca 960

gttttggagg gggacagagg agcttagggc ccaaggtgag gggctgggca gtgggcactt 1020

agccccagag gcagaggaag cagaggctcc aacctatgtc ggtatcccca ctggagtgag 1080

ctgcagacgg gaccttgttc attctgcctt ctgccatggg gatctgcctt tgaagggcaa 1140

tgggagaagt cctcctgggg actgcagctg tcgggggcag taccacatcg ggggaagagt 1200

gctcaaggca ggagctcttc ccgtcctgcc tggccactgg ctgccttgtg agccggacag 1260

gtggtccact gtgatggtta atgtccccct ccccacccac tcccagctac aggggtttct 1320

gataccatct gcgagccctg cccagtcggc ttcttctcca atgtgtcatc tgctttcgaa 1380

aaatgtcacc cttggacaag 1446

<210> 12

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cgcggtcgac aagctaagaa caaagtctgc ccttg 35

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ctaaaaacag aagtggacag 20

<210> 14

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cacttctgtt tttaggtcca tccagaacca cccac 35

<210> 15

<211> 62

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ctcttgctta ctgtcccttc tcgagtaaag caactgtctc gcttctgagt acgagtgtca 60

tg 62

<210> 16

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gacagtaagc aagagtatgt gtatgtgtgc atgtg 35

<210> 17

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

cgactctaga ggatccccca gctatcttct tactt 35

<210> 18

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

acctctccct aaccaatgac agg 23

<210> 19

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cctctcccta accaatgaca ggg 23

<210> 20

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cctgactttc cccaccccca tgg 23

<210> 21

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gactttcccc acccccatgg tgg 23

<210> 22

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ctcccaaaga cgcagcaggt agg 23

<210> 23

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ggtgaatgtg accttgtggc tgg 23

<210> 24

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

accttgtggc tggcttaagg 20

<210> 25

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

tgaccttgtg gctggcttaa ggg 23

<210> 26

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

cttagttaat agaagcctgt tgg 23

<210> 27

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

cccttttcag ttcactttct tgg 23

<210> 28

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

ttttcagttc actttcttgg tgg 23

<210> 29

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

tttcagttca ctttcttggt ggg 23

<210> 30

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

accaagaaag tgaactgaaa agg 23

<210> 31

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

ccaagaaagt gaactgaaaa ggg 23

<210> 32

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

caagaaagtg aactgaaaag ggg 23

<210> 33

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

ctttaagaag agacttcaag agg 23

<210> 34

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

gaagagactt caagaggata tgg 23

<210> 35

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

aagaggatat ggagaccaac agg 23

<210> 36

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

tgtcccttaa gccagccaca agg 23

<210> 37

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

caccagtaag tgacccacca tgg 23

<210> 38

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

gctaatacga ctcactatag ctcccaaaga cgcagcaggt gttttagagc tagaaatagc 60

aag 65

<210> 39

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

gctaatacga ctcactatag ccaagaaagt gaactgaaaa gttttagagc tagaaatagc 60

aag 65

<210> 40

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

aaaagcaccg actcggtgcc 20

<210> 41

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60

ggcaccgagt cggtgctttt 82

<210> 42

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

ttcctttcgc agaggtggtc t 21

<210> 43

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

ctcttgctta ctgtcccttc tcg 23

<210> 44

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

cagaagcgag acagttgctt tact 24

<210> 45

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

ttactttgtg gcactggtct aacc 24

<210> 46

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

ccctgtgatt tggctcttct 20

<210> 47

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

ggtgtctagg aattcgcttt ca 22

<210> 48

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

gaaggctggc actgtacg 18

<210> 49

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

cattagtctg actcgttcct ttct 24

<210> 50

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

acaaacagta cctccacgat g 21

<210> 51

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

ttcttaaccc gaagcccttg 20

Claims (10)

1. A construction method of humanized CD40 gene modified animal cells is characterized by comprising the following steps:

replacing the sequence of the No. 2 to No. 5 exons of the non-human CD40 gene in the non-human animal cell with the sequence of the No. 2 to No. 5 exons of the human CD40 gene to form a humanized CD40 gene, so as to obtain a humanized CD40 gene modified animal cell;

preferably, the non-human animal is a rodent; more preferably, the rodent is a mouse;

preferably, the cell is a fertilized egg.

2. The method for constructing humanized CD40 genetically modified animal cell according to claim 1, wherein the method for constructing humanized CD40 genetically modified animal cell comprises:

a mixture of a human CD40 gene homologous recombination vector, sgRNA, and Cas9, the Cas9 including Cas9mRNA and/or Cas9 protein, is provided and introduced into the non-human animal cell.

3. The method for constructing the humanized CD40 genetically modified animal cell according to claim 2, wherein the target site at the 5 'end of the sgRNA is located in intron 1 of mouse Cd40 gene, and the target site at the 3' end of the sgRNA is located in intron 5 of mouse Cd40 gene;

preferably, the sequence of the 5' end target site targeted by the sgRNA is as shown in SEQ ID NO: 18-27, the sequence of the sgRNA-targeted 3' end target site is as shown in SEQ ID NO: 28-37;

more preferably, the sequence of the 5' target site targeted by the sgRNA is as set forth in SEQ ID NO: 22, the sequence of the sgRNA-targeted 3' end target site is shown as SEQ ID NO: shown at 36.

4. The method for constructing humanized CD40 genetically modified animal cell according to claim 2, wherein the human CD40 gene homologous recombination vector comprises a 5 'homology arm, a human CD40 gene fragment and a 3' homology arm arranged in sequence from a 5 'end to a 3' end;

preferably, the sequence of the human CD40 gene fragment is shown in SEQ ID NO: 11 is shown in the figure;

preferably, the sequence of the 5' homology arm is as shown in SEQ ID NO: 9, the sequence of the 3' homology arm is shown as SEQ ID NO: shown at 10.

5. The method for constructing an animal cell transformed with humanized CD40 gene according to claim 1, wherein the humanized CD40 gene is selected from at least one of the following group:

(a) the CDS coding sequence of the humanized CD40 gene is shown as SEQ ID NO: 6 is shown in the specification;

(b) the mRNA sequence transcribed by the humanized CD40 gene is shown as SEQ ID NO: 7 is shown in the specification;

(c) the protein sequence coded by the humanized CD40 gene is shown as SEQ ID NO: shown in fig. 8.

6. A humanized CD40 genetically modified animal cell, wherein the humanized CD40 genetically modified animal cell is obtained by the method for constructing the humanized CD40 genetically modified animal cell according to any one of claims 1 to 5.

7. A method for constructing a humanized CD40 genetically modified animal model, which is characterized in that the humanized CD40 genetically modified animal cell of claim 6 or an embryo developed from the humanized CD40 genetically modified animal cell is transplanted into a surrogate mother body to be developed, so as to obtain an F0 generation animal;

f0 generation animals are tested to obtain positive animals with correct genotype identification of F0 generation.

8. The method of claim 7, wherein the method of constructing the humanized CD40 genetically modified animal model further comprises: and (3) expanding the population quantity of the F0 generation positive animals in a hybridization and selfing mode, selfing the heterozygote animals to obtain homozygote animals, and establishing a stable humanized CD40 gene modified animal strain.

9. A cell, cell line or cell culture, tissue or organ derived from a humanized CD40 genetically modified animal model or progeny thereof, wherein the humanized CD40 genetically modified animal model is obtained by the method of constructing the humanized CD40 genetically modified animal model of any one of claims 7-8.

10. Use of a humanized CD40 genetically modified animal model obtained by the method of constructing a humanized CD40 genetically modified animal model according to any one of claims 7 to 8, a cell, cell line or cell culture, tissue, organ derived from said humanized CD40 genetically modified animal model in the field related to the human CD40 gene or protein;

optionally, the application comprises at least one of human CD40 gene function research, human CD40 antibody research, medicine preparation aiming at a human CD40 target site and drug effect research.

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